Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions

Abstract

Streptomyces mobaraenesis transglutaminase can catalyze the cross-linking of proteins, which has been widely used in food processing. In this study, we rationally modified flexible regions to further improve the thermostability of FRAPD-TGm2 (S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L), a stable mutant of the transglutaminase constructed in our previous study. First, five flexible regions of FRAPD-TGm2 were identified by molecular dynamics simulations at 330 and 360 K. Second, a script based on Rosetta Cartesian_ddg was developed for virtual saturation mutagenesis within the flexible regions far from the substrate binding pocket, generating the top 18 mutants with remarkable decreases in folding free energy. Third, from the top 18 mutants, we identified two mutants (S116A and S179L) with increased thermostability and activity. Finally, the above favorable mutations were combined to obtain FRAPD-TGm2-S116A-S179L (FRAPD-TGm2A), exhibiting a half-life of 132.38 min at 60 °C (t 1/2(60 °C)) and a specific activity of 79.15 U/mg, 84 and 21% higher than those of FRAPD-TGm2, respectively. Therefore, the current result may benefit the application of S. mobaraenesis transglutaminase at high temperatures.

Efficient Production of a Thermostable Mutant of Transglutaminase by Streptomyces mobaraensis

“…However, these TGase mutants were only synthesized in E. coli in small amounts, , which are unsuitable for industrial production and application. In this study, S.…”

“…The thermal stability and catalytic activity of S. mobaraensis TGase have been significantly improved through molecular modification, ,,,, such as the FRAPD-TGm2 constructed in our previous study. However, these TGase mutants were only synthesized in E.…”

“…11,41 As the only commercial food-grade TGase, S. mobaraensis TGase was thermally unstable, which was the main obstacle to its industrial application. 42 The thermal stability and catalytic activity of S. mobaraensis TGase have been significantly improved through molecular modification, 7,11,12,42,43 such as the FRAPD-TGm2 12 constructed in our previous study. However, these TGase mutants were only synthesized in E. coli in small amounts, 12,43 which are unsuitable for industrial production and application.…”

“…42 The thermal stability and catalytic activity of S. mobaraensis TGase have been significantly improved through molecular modification, 7,11,12,42,43 such as the FRAPD-TGm2 12 constructed in our previous study. However, these TGase mutants were only synthesized in E. coli in small amounts, 12,43 which are unsuitable for industrial production and application. In this study, S. mobaraensis DSM40587 was subjected to ARTP mutation, 30 yielding the mutant smY2022 that can efficiently secrete TGase.…”

Rational Redesign of Chitosanase to Enhance Thermostability and Catalytic Activity to Produce Chitooligosaccharides with a Relatively High Degree of Polymerization

“…38 A growing body of research has shown that protein stability does not necessarily come at the expense of enzyme−substrate affinity and catalytic activity. 9,32,39 To some extent, local stiffness may be compatible with overall flexibility, and there is no contradiction between improving the overall stability of the protein and enhancing its affinity and catalytic efficiency. In this study, three software designs, based on multiple algorithms, were used to design mutants, and then, potential adverse mutations were screened out, based on the biophysical characteristics of the calculated three-dimensional structure of the mutant proteins.…”